The goal of the proposed work is to characterize in atomic detail sequence and structural elements that define HIV-1 immunogens. Using the technique of random systematic mutagenesis, we are able to generate libraries of chimeric human rhinoviruses (HRVs) that display HIV-1 sequences with many lengths, sequences, and conformations. The mutagenesis schemes have been designed to account for the large natural distribution of HIV-1 sequences. We are using immunoselection to isolate antigenic chimeras from "molecular haystacks". This system identifies chimeras that display HIV-1 epitopes in constrained conformations that are both antigenic and immunogenic. One of the HRV14:HIV-1 chimeras contains an HIV-1 sequence that does not match that of any known HIV-1 isolate (designated DN-6). This chimera elicited an anti-HIV-1 neutralizing response that is among the strongest reported for any AIDS vaccine system and was effective in neutralizing two of three HIV-1 strains tested. Three specific HRV14:HIV-1 libraries will be constructed: two focus on the HIV-1 V3 loop from gpl20 and one focuses on a conserved neutralizing epitope from gp41. A "cross-clade" V3 loop library is described that will contain composites of V3 loop sequences by encoding amino acid residues that exist in all clades. Another V3 loop library will focus on clade B sequences. A third library will involve insertion of the conserved gp41 "ELDKWA" immunogen in order to generate an immunodominant display of this sequence on the surface of HRV. Chimeric viruses with relevant HIV-1 antigenicity will be immunoselected from these libraries using neutralizing anti-HIV-1 antibodies. Immunoselected chimeras that are neutralized well by anti-HIV-1 antibodies with diverse specificities will be used to immunize guinea pigs and, in a number of cases, chimpanzees. Antisera elicited by HRV14:HIV-1 chimeras will be assayed for their ability to neutralize both laboratory-adapted and primary isolates of HIV- 1. We will determine the three-dimensional structures of a subset of the most immunogenic chimeras using X-ray crystallography. Two HRV14:HIV-1 chimeras (including DN-6) have been crystallized and diffract X-rays to at least 3.0 Angstroms resolution. The structures of these chimeras will be the first to reveal HIV-1 V3 loop sequences that are unquestionably in immunogenic conformations. A number of chimeras will be crystallized with Fab fragments of one or more anti-HIV monoclonal antibodies capable of neutralizing multiple strains of HIV-1. These structures will yield insights into structural determinants of immunogenicity and will hopefully aid in our understanding of how apparently similar sequences differ in the strength and specificity of the immune responses elicited. The information learned from these studies will be directly applicable to the design of useful vaccines against AIDS. A strength of the chimeric HRV:HIV system is that it can be used to present virtually any identified epitope (or mimotope thereof), identify viruses with immunological characteristics that mimic those of HIV, and enable the determination of the structures of the immunogenic HIV sequences at the atomic level.